Establishment of the basidiomycete Fomes fomentarius for the production of composite materials [original]

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

https://doi.org/10.1186/s40694-022-00133-y

RESEARCH

Establishment ofthebasidiomycete Fomes

fomentarius fortheproduction ofcomposite

materials

Carsten Pohl1† , Bertram Schmidt1† , Tamara Nunez Guitar1, Sophie Klemm2 , Hans‑Jörg Gusovius3 ,

Stefan Platzk4 , Harald Kruggel‑Emden4, Andre Klunker5 , Christina Völlmecke5 , Claudia Fleck2* and

Vera Meyer1*

Abstract

Background: Filamentous fungi of the phylum Basidiomycota are considered as an attractive source for the biotech‑

nological production of composite materials. The ability of many basidiomycetes to accept residual lignocellulosic

plant biomass from agriculture and forestry such as straw, shives and sawdust as substrates and to bind and glue

together these otherwise loose but reinforcing substrate particles into their mycelial network, makes them ideal can‑

didates to produce biological composites to replace petroleum‑based synthetic plastics and foams in the near future.

Results: Here, we describe for the first time the application potential of the tinder fungus Fomes fomentarius for lab‑

scale production of mycelium composites. We used fine, medium and coarse particle fractions of hemp shives and

rapeseed straw to produce a set of diverse composite materials and show that the mechanical materials properties

are dependent on the nature and particle size of the substrates. Compression tests and scanning electron microscopy

were used to characterize composite material properties and to model their compression behaviour by numerical

simulations. Their properties were compared amongst each other and with the benchmark expanded polystyrene

(EPS), a petroleum‑based foam used for thermal isolation in the construction industry. Our analyses uncovered that

EPS shows an elastic modulus of 2.37 ± 0.17 MPa which is 4‑times higher compared to the F. fomentarius composite

materials whereas the compressive strength of 0.09 ± 0.003 MPa is in the range of the fungal composite material.

However, when comparing the ability to take up compressive forces at higher strain values, the fungal composites

performed better than EPS. Hemp‑shive based composites were able to resist a compressive force of 0.2 MPa at 50%

compression, rapeseed composites 0.3 MPa but EPS only 0.15 MPa.

Conclusion: The data obtained in this study suggest that F. fomentarius constitutes a promising cell factory for the

future production of fungal composite materials with similar mechanical behaviour as synthetic foams such as EPS.

Future work will focus on designing materials characteristics through optimizing substrate properties, cultivation

conditions and by modulating growth and cell wall composition of F. fomentarius, i.e. factors that contribute on the

meso‑ and microscale level to the composite behaviour.

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Open Access

Fungal Biology and

Biotechnology

*Correspondence: claudia.fleck@tu‑berlin.de; vera.meyer@tu‑berlin.de

†Carsten Pohl and Bertram Schmidt shared first co‑authorship

1 Chair of Applied and Molecular Microbiology, Technische Universität

Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany

2 Chair of Materials Science and Engineering, Technische Universität

Berlin, Str. des 17. Juni 135, 10623 Berlin, Germany

Full list of author information is available at the end of the article

Page 2 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Introduction

Fungal biotechnology is an innovation driver for the bio-

economy with its principles of circular economy and sus-

tainability [1, 2]. Especially filamentous fungi have a rich

and very versatile metabolism that forms the basis for a

diverse palette of products, which become harnessed by

the food, beverage, pharmaceutical, biofuel, textile, feed,

automotive, packaging and chemical industries. How-

ever, filamentous fungi are not only masters of biosynthe-

sis, they are also masters of decomposition. Their ability

to degrade and transform lignocellulosic substrates into

composite materials is unique in nature and attracted a

lot of interest recently [1, 2]. In several interdisciplinary

endeavours, fungal bio(techno)logists, designers, process

engineers and material scientists have collaborated to

turn by-products from agriculture and forestry with the

help of basidiomycetes into composite materials as high-

lighted in recent reviews [3–5]. The vision is surprising

and fascinating, yet plausible and thus hopefully achiev-

able in the near future: Plastics, foams, textiles and other

materials derived from petroleum-based resources could

soon be functionally replaced by a new class of bioma-

terials produced by fungal biotechnology [2]. Given the

urgent need to reduce global carbon dioxide emission

and plastic pollution, the pressure to innovate is indeed

high. Within the last 5years, the ability of fungal myce-

lium not only to digest but also to bind and connect loose

plant-based particles into firmer composite materials

has thus led to a substantial increase in publications that

pioneered the manufacturing process and that described

some characteristics of mycelium-based materials [6–12].

Potential applications for fungal composite materials that

have been discussed so far are as diverse as disruptive—

soon packaging material, thermal insulation, acoustic

insulation, construction material as well as leather could

be produced by filamentous fungi of the phylum Basidi-

omycota [2–4, 13–15].

To contribute to these research efforts, we ran a bio-

prospecting program in 2018 in our Berlin-Brandenburg

area to explore the local biodiversity of mushroom-form-

ing fungi and to build up a strain collection of basidiomy-

cetes that reflects the predominant regional biodiversity

and that feeds well on regional renewable plant resources.

As recently described [16], we could isolate and identify

about 75 basidiomycetes, most of which were assigned

to the order Polyporales, including the tinder fungus

Fomes fomentarius, the fire sponge Phellinus robustus,

Ganoderma adspersum, the artist´s bracket Ganoderma

applanatum and the turkey tail Trametes versicolor. Also,

representatives of the order Agaricales became members

of the strain collection including the oyster mushroom

Pleurotus ostreatus, the stump mushroom Armillaria

ostoyae and the similar looking Pholiota limonella [16].

In growth experiments on different substrates from

regional agricultural residual streams, the white-rot fungi

F. fomentarius, P. ostreatus and T. versicolor excelled with

the best performance [16].

Various considerations let us to focus our further

research on the tinder fungus F. fomentarius. This basidi-

omycete, which is prevalent throughout the temperate

climate zone of the northern hemisphere, is well-known

to traditional medicine and thus has a rich ethnomyco-

logical tradition [17, 18]. Furthermore, the trama of its

fruiting bodies has been safely used by mankind for hun-

dreds of years as wound dressing and leather alternative

[19]. Remarkably, the fruiting bodies are water-repellent,

very stable and light-weighted. Interestingly, the hyme-

mium follows a hierarchically honeycomb structure and

was previously already subjected to mechanical test-

ing [20], showing compressive stress–strain curves of

foams, where an initially linear course is followed by an

extended plateau region [20]. Given that such charac-

teristics could be adjusted in the future for laboratory

cultivated F. fomentarius mycelia that were fed on renew-

able plant biomass, new materials for lightweight appli-

cations, specifically for anisotropic loading conditions

could be developed. Finally, the genome sequence of

one F. fomentarius isolate identified in France has been

recently published (strain CIRM-BRFM 1821) [21] and

uncovered many genes in its genome predicted to encode

lignin-active peroxidases and manganese peroxidases

which are key for the breakdown of lignin. As its genome

sequence contains less genes predicted to encode cellu-

lases, it grows less well on cellulose, which is typical for

white-rot fungi. F. fomentarius was thus recently ranked

with a moderate hyphal expansion rate on lignocellulosic

substrates but a high rate of decomposition of its sub-

strate when compared to another 20 basidiomycetes [22].

Another important premise for our decision was that F.

fomentarius grows well on local agricultural residues such

as hemp shives or rapeseed straw [16]. Hemp was once an

important source for fibres for the textile industry, but its

cultivation and use declined in the last century because

cotton and synthetic fibres became more popular. The

Keywords: Filamentous fungi, Fomes fomentarius, Circular economy, Bioeconomy, Hemp, Rapeseed, Composite

material, Mycelium, Neo‑Hooke model, Finite element method FEM, Mechanical properties, Compressive strength,

Stiffness

Page 3 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

worldwide annual area of hemp harvested mainly for

seeds and fibres is reported with about 150,000ha for

2018 [23]. In Europe alone, the area under cultivation

has increased to over 40,000ha in recent years, which

represents a potential use for at least 60,000 t of shives

[23]. Hemp fibres currently experience a resurgence of

interest by the textile industry as an environmentally

friendly alternative to cotton, the cultivation of which is

high in water demand, pesticide use and soil salinization

[23]. In contrast, hemp is a frugal but high-yielding plant

that has no pesticide and low fertilizer demand but uses

water about six times more efficiently for biomass forma-

tion than cotton [24, 25]. Thus, hemp can grow well even

under hot and dry conditions and on poor-soil sites such

as prevalent in the Berlin-Brandenburg area and beyond

[26]. The second main product after hemp fibre separa-

tion, the shives, are currently very often under-valued in

applications like animal bedding. But with its content of

about 48% w/w cellulose, 21 to 25% w/w hemicellulose

and 17–19% w/w lignin [27], hemp shives are ideal sub-

strates for both white-rot and brown-rot basidiomycetes.

Rapeseed will remain an important source of oil pro-

duced for food and feed as well as technical use, although

it has a high water and fertilizer demand and the land use

efficiency can be regarded as critical in terms of biodiesel

production due to the low energy efficiency [28]. The

Food and Agriculture Organization of the United Nations

lists the harvested area of rapeseed as 36.96 Mio ha. In

Brandenburg, winter rapeseed is the most important oil-

seed crop with an acreage of about 77,000 ha [29], which

takes up about 10% of the arable land [30]. The composi-

tion of rapeseed straw is very similar to hemp shives with

about 37% w/w cellulose, 24% w/w hemicellulose and

about 17% w/w lignin [31] and thus well suited as a sub-

strate for both white-rot and brown-rot basidiomycetes.

In the current study, we describe the cultivation of F.

fomentarius on both hemp shives and rapeseed straw

for the production of composite materials. We applied

compression tests to determine the compressive Young’s

Modulus as recently described for composite materials

obtained with Schizophyllum, Ganoderma and Trametes

species, respectively [7, 32, 33] and used scanning elec-

tron microscopy to characterize the composite structure

and mechanical properties. We used the experimental

data for numerical simulations of the compression behav-

iour. We furthermore studied the impact of the substrate’s

particle sizes on the composite material properties and

used fine, medium and coarse fractions of hemp shives

and rapeseed straw to produce a set of diverse composite

materials. Their properties were compared amongst each

other and with the benchmark expanded polystyrene

(EPS), a petroleum-based foam used for thermal isolation

in the construction industry.

Results anddiscussion

Substrate preparation andclassification

The particle size of both hemp shives and rapeseed straw

substrates were reduced by means of a laboratory cutting

mill. To estimate the mass percentages of the subsequent

classification products, sieve analyses of the milling prod-

ucts were carried out using analytical sieves and shakers.

Rapeseed straw showed significantly larger amounts of

screening residue of mesh sizes above 8mm (Additional

file1). Furthermore, fine fractions below 0.63mm mesh

size were found at about 5% weight fraction. To achieve

a mass distribution of approximately one third each for

small, medium, and large fraction, the results suggested

classification cut sizes of 2mm and 3.15mm, given the

available screens. Consequently, these mesh sizes were

utilized during the following classification processes via

a Mogensen Sizer. Simultaneously, a 0.65mm screen was

used with the intention to reduce the finest particles such

as dust. Hence, three particle fractions were prepared for

each substrate: small (0.63–2mm), medium (2–3.15mm)

and large (> 3.15mm–6.3mm). The resulting mass per-

centages are shown in Fig.1.

Fig. 1 Mass distribution of plant substrate fractions after classification

Page 4 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

Finally, bulk density was determined for all fractions

by measuring the total bulk volumes of the fractions

(Additional file2). Each fraction was subjected to further

analysis with respect to particle size and shape by means

of digital image analysis. A significantly larger number

of finest particles were found within all rapeseed straw

samples compared to hemp shives, possibly due to differ-

ences in abrasion resistance (note that results from image

analysis are based on the number of particles rather than

mass percentages). In Fig.2, histograms of the ratios of

minimum to maximum Feret diameters of hemp shives

and rapeseed straw middle fractions are depicted. While

the rapeseed straw’s modal value is smaller than the cor-

responding value for hemp shives, its distribution is

broader and leans towards larger Feret ratios. Within the

examined fraction, hemp shives display thinner and more

elongated shapes.

Cultivation ofF. fomentarius andmanufacturing

ofcomposite materials

F. fomentarius grows well on malt extract agar (MEA),

glucose-based complete medium (CM) and on lignocel-

lulosic substrates such as hemp shives and forms hyphae

with a mean diameter of 2.8µm (n = 300, SD = 0.7, Fig.3).

A three-stage laboratory manufacturing process was

established for F. fomentarius (for details see “Methods”

section). In the first stage, mycelium harvested from malt

agar plates (Fig.4A) was used to inoculate millet grains

Fig. 2 Histograms of minimum to maximum Feret diameter ratios for hemp shives (left) and rapeseed straw (right)

Fig. 3 A F. fomentarius colonies after incubation at 25 °C in the dark for 96 h and 186 h. Doubling time of colony surface area on MEA and CM are

6 h (n = 8, SD = 1) and 14 h (n = 8, SD = 2) respectively. As the fungus also grows into the shives and towards the bottom of the agar plate, it is

impossible to estimate a doubling time based on radial growth measurement when cultivated on hemp shives inoculated with pure F. fomentarius

mycelium or with millet spawn. B Light microscopic images of F. fomentarius hyphae when cultivated in liquid CM for 96 h and 186 h, respectively at

400× magnification

Page 5 of 13

Pohletal. Fungal Biology and Biotechnology (2022) 9:4

to obtain precultures of F. fomentarius during a 2-week

cultivation (Fig.4B). This ‘millet spawn’ then served as

inoculum to inoculate 3-L bag cultures of hemp shives

and rapeseed straw for the second stage cultivation. For

future industrial upscaling efforts, however, we propose

that the millet preculture should be substituted by non-

food plant substrates that become mixed with non-inoc-

ulated plant substrates to avoid extensive use of cereal

grains. After the 2-week cultivation in substrate bags, the

overgrown substrates were shred and transferred into

sterile cylindrical moulds (Fig.4C and D), to allow for a

final cultivation with the duration of 2weeks, whereby

the moulds were removed after one week (Fig.4E). For

each condition tested (substrate, particle fraction), at

least six biological replicates were produced. The final

composite materials obtained with this manufacturing

process were optically inspected after cutting, revealing

a gradient of fungal growth within the test specimens

Fig. 4 Laboratory manufacturing process for F. fomentarius composite materials. A Inoculation of sterile millet with for F. fomentarius mycelium

followed by an incubation for 2 weeks at 25 °C in the dark. B Inoculation of hemp shives (or rapeseed straw) cultivation bags with the millet spawn

followed by an incubation for 1 week at 25 °C in the dark. Note that the use of millet spawn for inoculation has the advantage of good mixing

properties in the 3‑L cultivation bags used and thus generation of more homogeneous growth throughout the plant substrates. C Shredding of

preliminary hemp shives (or rapeseed straw) composites and transfer of the material into moulds. D Filled moulds before cultivation for 1 week at

25 °C in the dark. E Sample appearance after 1 week of cultivation. Moulds are removed to allow thorough overgrowth of the samples for another

week. F Drying in an oven at 60 °C for 2 days and final appearance of composites used for compression tests

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